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Supplementary Figures S1-S3.

Purpose: Primary Waldenstrom's Macroglobulinemia (WM) cells present with a significantly higher level of the immunoproteasome compared with the constitutive proteasome. It has been demonstrated that selective inhibition of the chymotrypsin-like (CT-L) activity of constitutive-(c20S) and immuno-(i20S) proteasome represents a valid strategy to induce antineoplastic effect in hematologic tumors. We therefore evaluated carfilzomib, a potent selective, irreversible inhibitor of the CT-L activity of the i20S and c20S in WM cells.Experimental Design: We tested the effect of carfilzomib on survival and proliferation of primary WM cells, as well as of other IgM-secreting lymphoma cell lines. Carfilzomib-dependent mechanisms of induced apoptosis in WM cells, and its effect on WM cells in the context of bone marrow (BM) microenvironment have been also evaluated. Moreover, the combinatory effect of carfilzomib and bortezomib has been investigated. In vivo studies have been performed.Results: We demonstrated that carfilzomib targeted the CT-L activity of both i20S and c20S, which led to the induction of toxicity in primary WM cells, as well as in other IgM-secreting lymphoma cells. Importantly, carfilzomib targeted WM cells even in the context of BM milieu. In addition, carfilzomib induced apoptosis through c-jun-N-terminal-kinase activation, caspase cleavage, and initiation of unfolded protein response. Importantly, the combination of carfilzomib and bortezomib synergistically inhibited CT-L activity, as well as caspase-, PARP-cleavage and GRP94 expression. Antitumor activity of carfilzomib has been validated in vivo.Conclusions: These findings suggest that targeting i20S and c20S CT-L activity by carfilzomib represents a valid antitumor strategy in WM and other IgM-secreting lymphomas. Clin Cancer Res; 17(7); 1753–64. ©2011 AACR.

Objectives To explore health behavior profiles of AI/AN youth involved in native students together against negative decisions (STAND), a national culture-based curriculum. Methods We analyzed data from 1236 surveys conducted among AI/AN youth at 40 native STAND implementation sites located in 16 states throughout the US from 2014 to 2017. Health profiles included demographics, sexual orientation, sexual activity, STI testing, cigarette use, and suicide attempts in the past 12-months. We used t-tests and chi square tests of independence to compare risk behavior prevalence among the sample. Results Health behavior profiles of AI/AN youth indicate that 45.6% of youth did not use condoms the last time they had sex, and 82.7% have never been tested for STIs. Differences in cigarette smoking were observed in questioning youth (questioning: 80.3%, straight/heterosexual: 63.8%, LGBTQ2S + : 49.9%, p = 0.03). Conclusions for Practice Health behaviors related to sex, substance, violence and self-harm, are at least as common for AI/AN youth as those observed in other US teens. Future research should consider similarities and differences in health profiles of AI/AN youth when designing interventions that affect them. Further, our findings underscore the need for culturally-relevant curricula like native STAND, not because their health behavior is different, but because their socio-ecologic environment is different.

Culturally-adapted evidence-based programs (EBPs) are needed to promote healthy behaviors among Native teens and young adults. Little is known about the facilitators and barriers of implementing and sustaining EBPs in Native communities. This paper aims to identify those factors described by educators who implemented the Native Students Together Against Negative Decisions (STAND) curriculum. Methods. We conducted qualitative, semi-structured interviews with 44 Native STAND educators from 48 sites throughout the United States. We used a modified grounded theory approach to explore barriers, facilitators, and sustainability factors related to implementing Native STAND. Results. We learned that disruptions to staffing, coordination, and organizational factors were the most common barriers. Factors that improved implementation success included: tailoring the program to local needs/constraints, having a supportive Project Manager, improved fidelity due to check-in calls, and participation in summer training. Factors that improved sustainability included: access to needed infrastructure, administrative support, community support, and student interest. Discussion. The delivery of Native STAND was further improved by person-to-person communication and resource sharing across sites. Sustaining EBPs in AI/AN settings requires culturally-tailored technical assistance, sufficient implementation funds for materials and staffing, and a community of peer educators to inspire forward progress. Conclusion. EBPs that reflect the needs and experiences of American Indian and Alaska Native (AI/AN) youth are necessary to address systemic inequities in adolescent health outcomes. The Native STAND Dissemination and Implementation study is among the first to assess facilitators and barriers to program delivery in diverse AI/AN settings.

We evaluated a telehealth training curriculum to prepare Registered Behavior Technicians® (RBTs®) to conduct caregiver coaching on behavior analytic skills via telehealth.A non-concurrent multiple baseline across therapists design was used to evaluate the training procedures delivered by Board Certified Behavior Analysts® (BCBAs®). All therapists (RBTs) worked with one family during the baseline and training phases and an additional session was conducted with a different family during the novelty probe condition to see if therapists were able to perform the newly acquired skills without BCBA support.Seven therapists who received behavioral skills training on the curriculum reached mastery criteria within three sessions. All RBTs were able to provide caregiver coaching via telehealth with high degrees of fidelity and were able to independently conduct a telehealth session with a new family without support from the BCBA.Results of the current study provide support for the use of the telehealth training curriculum to prepare RBTs to conduct ABA services via telehealth.

Culturally relevant interventions have the potential to improve adolescent health and protective factors associated with sexual risk taking. We evaluated the impact of the Native STAND curriculum with American Indian and Alaska Native (AI/AN or Native) high school students living across the U.S. using a pre-post evaluation design. Native STAND is a comprehensive sexual health curriculum for Native high school students that focuses on sexually transmitted infections, HIV/AIDS, and teen pregnancy prevention, while also covering drug and alcohol use, suicide, and dating violence. The curriculum was implemented in 48 AI/AN communities from 2014 to 2019. A significantly higher percentage of youth reported at post-test having a serious conversation about sex with their friends (post 36% vs. pre 28%, p &lt, 0.001), thinking about lessons learned (post 24% vs. pre 7%, p &lt, 0.0001), and sharing lessons learned during the conversation (post 21% vs. pre 4%, p &lt, 0.001). A lower percentage of AI/AN youth reported being bullied in the last year (post 31% vs. pre 37%, p &lt, 0.001). Family social support was moderated by dose, with subscale scores of 3.75 at post-with &lt, 27 sessions vs. 3.96 at post-with all 27 sessions (p = 0.02). The results demonstrate the effectiveness of Native STAND when delivered in a variety of settings. Efforts are now underway to update Native STAND for medical accuracy, improve alignment with typical class periods, and promote its use and an effective EBI for AI/AN youth.

We performed a calendar-matched, 12-month, before (November 27, 2017 to November 26, 2018) and after (November 27, 2018 to November 26, 2019) study, to assess the utility of an emergency department-based HIV screening program. There were 710 and 14 335 patients screened for HIV during the pre and post-best practice alert (BPA) periods, respectively, representing more than a 20-fold increase in HIV screening following BPA implementation. Total HIV positive tests increased 5-fold following BPA implementation.

American Indian and Alaska Native (AI/AN) youth are strong in culture and rich in heritage and experience unique strengths and challenges throughout adolescence. Documenting conditions that protect against risk factors associated with poor health outcomes are needed. We explored scales that measure self-esteem, culture, social support, and community from a sample of 1,456 youth involved in Native STAND, a culturally-relevant evidence-based sexual health intervention. We established content validity by reviewing existing literature and community feedback. Construct validity was examined using factor analysis. The final self-esteem model included seven items, factor loadings ranged from 0.47 to 0.63 for positive self-esteem and 0.77 to 0.81 for negative self-esteem. The final culture model included three items, factor loadings 0.73 to 0.89. The social support scale included four items, factor loadings ranged from 0.86 to 0.87 for family social support and 0.75 to 0.77 for friends social support. The community and community safety scale included three items; factor loadings ranged from 0.52 to 0.82. Coefficient alphas for scales ranged from α = 0.63 to α = 0.86. This study validated scales in a national sample of AI/AN youth–psychometric scales provide an essential tool for documenting the needs and strengths of AI/AN youth.

Safe prescribing for persons with severe mental illness requires laboratory monitoring for psychotropic drug levels and metabolic side effects. Barriers to appropriate and timely monitoring increase when clients must obtain phlebotomy services at a separate facility. This quality improvement project was conducted within a program for assertive community treatment (PACT). Specific aims were to increase access to laboratory testing, improve efficiency, and lower costs by implementing on-site specimen collection. Outcomes, measured three months post-implementation, indicate that over half of all labs were obtained on-site, clients and staff were pleased with increased efficiencies, and costs were reduced by 37%.

Objective: We evaluated the effect of a behaviorally enhanced quality improvement intervention in reducing the number of antibiotic prescriptions written for antibiotic nonresponsive acute respiratory infections (ARIs). A secondary objective was identifying whether a reduction in inappropriate antibiotic prescriptions, if present, persisted after the immediate implementation of the intervention. Design: Nonrandomized, quasi-experimental study conducted from January 2017 through February 2020. Setting: University of California, Davis Health outpatient clinics. In total, 21 pediatric, family, and internal medicine practices in 10 cities and towns were included. Patients: Patients evaluated by a participating physician at an enrolled practice site during the study period with diagnoses (primary and secondary) from the International Classification of Diseases, Tenth Revision codes consistent with antibiotic nonresponsive ARI diagnoses. Intervention: A behaviorally enhanced quality improvement intervention to reduce inappropriate prescribing for antibiotic nonresponsive ARI. Results: In total, 63,028 eligible patient visits across 21 locations were included in the analysis. The most frequently prescribed antibiotic for antibiotic nonresponsive ARI was azithromycin (n = 3,551), followed by amoxicillin (n = 924). Overall, the intervention was associated with an immediate 46% reduction in antibiotic prescriptions for antibiotic nonresponsive ARI (P = .001) following the intervention. We detected no significant change in the month-to-month trend after the intervention was implemented (P = .87), indicating that the reduction was sustained throughout the postintervention period. Conclusion: Our findings demonstrate that a behaviorally enhanced quality improvement intervention to reduce inappropriate prescribing for antibiotic nonresponsive ARI in ambulatory care encounters was successful in reducing potentially inappropriate prescriptions for presumed antibiotic nonresponsive ARI.

BackgroundThe prevalence of syphilis is increasing in many countries, including the USA. The ED is often used by underserved populations, making it an important setting to test and treat patients who are not evaluated in outpatient clinical settings. We aimed to assess the utility of an ED-based syphilis and gonorrhoea/chlamydia cotesting protocol by comparing testing practices before and after its implementation.MethodsWe implemented an electronic health record (EHR) alert that prompted clinicians to order syphilis testing in patients undergoing gonorrhoea/chlamydia testing. We performed a retrospective cohort analysis that compared outcomes between the preimplementation period (January–November 2018) and the postimplementation period (January–November 2019). Patients were tested for Treponema pallidum antibody (TPA) using a multiplex flow immunoassay (MFI), and positive results were confirmed by rapid plasma reagin (RPR). The primary implementation outcome was the number of syphilis tests/month, and the primary clinical outcome was the number of syphilis diagnoses/month (defined as positive TPA MFI and RPR). We performed an interrupted time-series analysis to evaluate the effect of implementing the alert over time.ResultsFour-hundred and ninety-four and 1106 unique patients were tested for syphilis in the preimplementation and postimplementation periods, respectively. Syphilis testing increased by 55.6 tests/month (95% CI 45.9 to 65.3, pConclusionsOur study demonstrates that use of a targeted EHR alert testing protocol can increase syphilis testing and diagnosis and may reduce clinician bias in testing.

Objectives: Tight hamstrings contribute to inefficiency of movement and increased risk for injury. Static stretching is the most common intervention for this problem, but the use of alternatives like instrument-assisted soft tissue mobilization (IASTM) and proprioceptive neuromuscular facilitation (PNF) is increasing among clinicians. This study examined two prospective studies with the common aim of demonstrating the effectiveness of IASTM or PNF over static stretching for improving hamstring tightness. Methods: Nondisabled adults were recruited on a university campus. IASTM study: N = 17 (11 males and 6 females). PNF study: N = 23 (7 males and 16 females). Hip flexion range of motion was measured with a passive straight leg raise (for IASTM) or active straight leg raise (for PNF) before and after stretching. Participants performed a self-static stretch on one leg and received the alternative intervention on the contralateral leg. The two studies were analyzed separately for reliability indices and significant differences between interventions. Results: Hip flexion measures showed good reliability in both studies (intraclass correlation coefficient = 0.97) with a minimal detectable change of

Excess uric acid has been shown to induce oxidative stress, triglyceride accumulation, and mitochondrial dysfunction in the liver and is an independent predictor of type-2 diabetes. Skeletal muscle plays a dominant role in type 2 diabetes and presents a large surface area to plasma uric acid. However, the effects of uric acid on skeletal muscle are underinvestigated. Our aim was therefore to characterize the effects of excessive uric acid on oxidative stress, triglyceride content, and mitochondrial function in skeletal muscle C2C12myotubes and assess how these are modulated by the antioxidant molecule melatonin. Differentiated C2C12myotubes were exposed to 750 µM uric acid or uric acid + 10 nM melatonin for 72 h. Compared with control, uric acid increased triglyceride content by ~237%, oxidative stress by 32%, and antioxidant capacity by 135%. Uric acid also reduced endogenous ROUTINE respiration, complex II-linked oxidative phosphorylation, and electron transfer system capacities. Melatonin counteracted the effects of uric acid without further altering antioxidant capacity. Our data demonstrate that excess uric acid has adverse effects on skeletal muscle similar to those previously reported in hepatocytes and suggest that melatonin at a low physiological concentration of 10 nM may be a possible therapy against some adverse effects of excess uric acid.NEW & NOTEWORTHY Few studies have investigated the effects of uric acid on skeletal muscle. This study shows that hyperuricemia induces mitochondrial dysfunction and triglyceride accumulation in skeletal muscle. The findings may explain why hyperuricemia is an independent predictor of diabetes.

Glioma constitutes the most common type of primary brain tumor with a dismal survival, often measured in terms of months or years. The thin line between treatment effectiveness and patient harm underpins the importance of tailoring clinical management to the individual patient. Randomized trials have laid the foundation for many neuro-oncological guidelines. Despite this, their findings focus on group-level estimates. Given our current tools, we are limited in our ability to guide patients on what therapy is best for them as individuals, or even how long they should expect to survive. Machine learning, however, promises to provide the analytical support for personalizing treatment decisions, and deep learning allows clinicians to unlock insight from the vast amount of unstructured data that is collected on glioma patients. Although these novel techniques have achieved astonishing results across a variety of clinical applications, significant hurdles remain associated with the implementation of them in clinical practice. Future challenges include the assembly of well-curated cross-institutional datasets, improvement of the interpretability of machine learning models, and balancing novel evidence-based decision-making with the associated liability of automated inference. Although artificial intelligence already exceeds clinical expertise in a variety of applications, clinicians remain responsible for interpreting the implications of, and acting upon, each prediction.

Background Up to 44% of women who experience a urinary tract infection (UTI) develop a recurrent infection (rUTI) within one year. Insufficient evidence about risk factors for recurrence limits clinicians’ ability to identify women at risk for rUTI who may benefit from further interventions. Methods We conducted a retrospective cohort study of women age ≥18 years who presented for treatment of a UTI at Oregon Health & Science University ambulatory care clinics between 2011 and 2016. Pregnant women as well as those with a recent urinary catheter, genitourinary (GU) procedure, or hospitalization were excluded. The outcome was defined as the first episode of rUTI within one year of an index UTI. Demographics, biologically relevant comorbidities, and the antibiotic prescribed to treat the index UTI were evaluated as potential risk factors for first rUTI using multivariable logistic regression. A best subsets approach was used to determine the most parsimonious model. Results A total of 3,632 patients met inclusion criteria. The mean age of the cohort was 50 ± 20 years and 12% had a diabetes diagnosis. To treat their index UTI, 36% of women were prescribed fluoroquinolones, 33% sulfamethoxazole and/or trimethoprim, and 25% nitrofurantoin. Over the study period, the cumulative incidence of first rUTI was 16% (95% confidence interval (CI): 15.3%, 17.7%); 35% (95% CI: 31%, 39%) of these patients had >1 rUTI. Our model identified age (Odds ratio (OR): 1.02; 95% CI: 1.01, 1.02), urban residence (OR: 1.78; 95% CI: 1.28, 2.57), and neurologic disease diagnosis (OR: 1.46; 95% CI: 1.13, 1.89) affecting GU function (e.g., multiple sclerosis or spinal cord injury) as significant, independent risk factors of first rUTI after adjusting for the confounding effects of diabetes, obesity, and history of stroke or other cerebrovascular disease. Conclusion Diagnosis of neurologic disease that impacts GU function, age, and urban residence were identified as significant risk factors for first rUTI. The antibiotic selected to treat patients’ index UTI was not a significant risk factor for first rUTI. Future studies are needed to identify risk factors beyond what is currently captured discretely in the electronic health record to address critical gaps in our understanding of risk factors for rUTI. Disclosures J. C. McGregor, Merck: Grant Investigator, Research grant

Purpose: There is no standard of therapy for the treatment of Waldenström macroglobulinemia (WM), therefore there is a need for the development of new agents. Fibroblast growth factor receptor 3 (FGFR3) was shown to play a major role in several types in cancer. Dovitinib, an inhibitor of FGFR3, was effective in hematologic malignancies. In this study, we tested FGFR3 as a therapeutic target in WM and tested the effect of dovitinib on cell proliferation and apoptosis of WM cells in the context of BM microenvironment. Methods: The expression of FGFR3 in WM cells was tested using immunofluorescence and flow cytometry. Cell signaling in response to stimulation with FGF3 and stromal cells, and its inhibition by dovitinib was performed using immunoblotting. Cell survival and cell proliferation were assessed by MTT and BrdU assays. Apoptosis was measured by detection of APO-2.7 and cleavage of caspase-3 using flow cytometry. Cell cycle was performed by PI staining of cells and flow cytometry. The combinatory effect of dovitinib with other drugs was analyzed using Calcusyn software. The effect of dovitinib was tested in vivo. Results: FGFR3 was overexpressed in WM cells and its activation induced cell proliferation. Inhibition of FGFR3 with dovitinib decreased cell survival, increased apoptosis, and induced cell cycle arrest. Inhibition of FGFR3 by dovitinib reduced the interaction of WM to bone marrow components, and reversed its proliferative effect. Dovitinib had an additive effect with other drugs. Moreover, dovitinib reduced WM tumor progression in vivo. Conclusion: We report that FGFR3 is a novel therapeutic target in WM, and suggest dovitinib for future clinical trial the treatment of patients with WM. Clin Cancer Res; 17(13); 4389–99. ©2011 AACR.

Non-steroidal anti-inflammatory drugs (NSAIDs) are known to inhibit prostaglandin synthetic enzyme, cyclooxygenases (COXs), as well as to exhibit anti-tumor activity although at much higher concentrations. 15-Hydroxyprostaglandin dehyrogenase (15-PGDH), a key prostaglandin catabolic enzyme, was recently shown to be a tumor suppressor. Effects of NSAIDs on 15-PGDH expression were therefore examined. Flurbiprofen and several other NSAIDs were found to induce 15-PGDH expression in human colon cancer HT29 cells. Flurbiprofen, the most active one, was also shown to induce 15-PGDH expression in other types of cancer cells. Induction of 15-PGDH expression appeared to occur at the stage of mRNA as levels of 15-PGDH mRNA were increased by flurbiprofen in HT29 cells. Levels of 15-PGDH were also found to be regulated at the stage of protein turnover. MEK inhibitors, PD98059 and U-0126, which inhibited ERK phosphorylation were shown to elevate 15-PGDH levels very significantly. These inhibitors did not appear to alter 15-PGDH mRNA levels but down-regulate matrix metalloproteinase-9 (MMP-9). This protease was shown to degrade and inactivate 15-PGDH suggesting that elevation of 15-PGDH levels could be due to inhibition of MMP-9 expression by these inhibitors. Similarly, flurbiprofen was also demonstrated to inhibit ERK activation and to down-regulate MMP-9 expression. Furthermore, flurbiprofen was shown to induce the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1), an inhibitor of MMP-9. The turnover of 15-PGDH was found to prolong in the presence of flurbiprofen as compared to that in the absence of this drug. Taken together, these results indicate that flurbiprofen up-regulates 15-PGDH by increasing the expression and decreasing the degradation of 15-PGDH in HT29 cells.

Oxidation of fatty acids is a major source of energy in the heart, liver, and skeletal muscle. It can be measured accurately using respirometry in isolated mitochondria, intact cells, and permeabilized cells or tissues. This technique directly measures the rate of oxygen consumption or flux at various respiratory states when appropriate substrates, uncouplers, and inhibitors are used. Acylcarnitines such as palmitoylcarnitine or octanoylcarnitine are the commonly used substrates. The β-oxidation pathway is prone to feedforward inhibition resulting from accumulation of short-chain acyl-CoA and depletion of CoA, but inclusion of malate or carnitine prevents accumulation of these intermediaries and CoA depletion.

The widespread involvement of the bone marrow with tumor cells indicates that there is continuous cell trafficking of WM cells in and out of the bone marrow leading to cell dissemination in the bone marrow and in the lymph nodes in many patients with WM. The interaction of the WM cells with the bone marrow is critical for the regulation of cell proliferation, cell cycle, drug resistance as well as cell dissemination and trafficking. Advances in understanding the interaction of the tumor clone with the BM microenvironment have led to the development of therapeutic agents that not only target the tumor clone but also regulate the bone marrow microenvironment. Here, we review the role of the cellular and liquid bone marrow compartments in the regulation of cell proliferation and dissemination in WM.

Epigenetics represent heritable changes in gene expression that are not due to any alteration in the DNA sequence. One of the best-known epigenetic markers is histone acetylation, which has been shown to be deregulated in neoplastic diseases, including B-cell malignancies, such as Waldenström's Macroglobulinemia (WM), a low-grade B-cell lymphoma characterized by the presence of lymphoplasmacytic cells in the bone marrow and a serum monoclonal immunoglobulin M in the circulation. It has been recently demonstrated that microRNAs may be responsible for modulating histone acetylation in WM cells, thus providing the preclinical evidences for using microRNA-based therapeutic strategies in this disease.

Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment to regulate multiple cellular processes. Rapamycin and its analogs have not shown significant activity in multiple myeloma (MM), likely because of the lack of inhibition of TORC2. In the present study, we investigated the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. TORC1/2 knock-down led to significant inhibition of the proliferation of MM cells, even in the presence of BM stromal cells. We also tested INK128, a dual TORC1/2 inhibitor, as a new therapeutic agent against these MM cell lines. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin), even in the presence of cytokines or stromal cells. In vitro and in vivo studies showed that p-4EBP1 and p-Akt inhibition could be predictive markers of TORC2 inhibition in MM cell lines. Dual TORC1/2 inhibition showed better inhibition of adhesion to BM microenvironmental cells and inhibition of homing in vivo. These studies form the basis for further clinical testing of TORC1/2 inhibitors in MM.

Purpose: The ephrin receptors (Eph) are found in a wide range of cancers and correlate with metastasis. In this study, we characterized the role of Eph-B2 receptor in the interaction of Waldenstrom's macroglobulinemia (WM) cells with the bone marrow microenvironment. Experimental Design: We screened the activity of different receptor tyrosine kinases in WM patients and found that Eph-B2 was overexpressed compared with control. Also, we tested the expression of ephrin-B2 ligand on endothelial cells and bone marrow stromal cells (BMSC) isolated from WM patients. We then tested the role of Eph-B2/Ephrin-B2 interaction in the adhesion of WM cells to endothelial cells and BMSCs; the cell signaling induced by the coculture in both the WM cells and the endothelial cells; WM cell proliferation, apoptosis, and cell cycle in vitro and tumor progression in vivo; and in angiogenesis. Results: Eph-B2 receptor was found to be activated in WM patients compared with control, with a 5-fold increase in CD19+ WM cells, and activated cell adhesion signaling, including focal adhesion kinase, Src, P130, paxillin, and cofilin, but decreased WM cell chemotaxis. Ephrin-B2 ligand was highly expressed on endothelial cells and BMSCs isolated from WM patients and on human umbilical vein endothelial cells and induced signaling in the endothelial cells promoting adhesion and angiogenesis. Blocking of ephrin-B2 or Eph-B2 inhibited adhesion, cytoskeletal signaling, proliferation, and cell cycle in WM cells, which was induced by coculture with endothelial cells and decreased WM tumor progression in vivo. Conclusion: Ephrin-B2/Eph-B2 axis regulates adhesion, proliferation, cell cycle, and tumor progression in vivo through the interaction of WM with the cells in the bone marrow microenvironment. Clin Cancer Res; 18(1); 91–104. ©2011 AACR.

Waldenstrom macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by widespread involvement of the bone marrow with lymphoplasmacytic cells. In approximately 20% of patients, the malignant clone also involves the lymph nodes and induces hepatosplenomegaly. The mechanisms by which the tumor cells home to the bone marrow and preferentially reside in the marrow niches are not fully elucidated. In this review, we examine the role of the bone marrow microenvironment in the regulation of cell growth, survival and cell dissemination in WM. We also summarize specific regulators of niche-dependent tumor proliferation in WM. These include chemokines, adhesion molecules, Src/PI3K/Akt/mTOR signaling, NF-kB activation, and micro-RNA regulation in WM. Targeting these pathways in clinical trials could lead to significant responses in this rare disease.

Abstract 2424 Introduction Tumor necrosis factor receptor super families (TNFRSFs) play an important role in activation of lymphocyte and cell apoptosis. However the function of TNFRSFs in multiple myeloma (MM) remains unknown. Loss of function mutation of Fas antigen (TNFRSF6) was identified in MM cells, thus suggesting the possible role of TNFRSFs in regulating MM pathogenesis. We therefore investigated the epigenetic mechanisms that may mediate inactivation of TNFRSFs and its functional role in MM. Methods Dchip software was utilized for analyzing gene expression dataset. DNA was extracted from both primary CD138+ MM plasma cells and MM cell lines using blood & tissue DNA isolation kit (Qiagen, Inc.). Expression of GITR in primary CD138+ plasma cells was detected by Imunohistochemistry (IHC) DNA methylation was analyzed by methylated DNA immunoprecipitation (Medip) assay and bisulfate sequencing. 5'azacytidine was used to demethylate genomic DNA. Gene expression was detected by qRT-PCR and confirmed at the protein level by flow cytometry and western-blot. Over-expression of GITR was obtained in MM1.S cells by using GITR recombinant plasmid and electroporation. Apoptosis was determined using Annexin/PI staining and flow cytometry analysis. Activation of apoptotic signaling was studied by western blot. Cell survival and proliferation were analyzed by MTT and BrdU assay, respectively. Recombinant GITR-lentivirus was obtained from the supernatant of culture medium after 72 hours transfection in 293 cells. GFP positive MM cells were sorted and analyzed by flow cytometry. In vivo effect of GITR on MM tumor growth was determined by injection of GITR over-expressing MM cells in null mice. Mice skull, femur and vertebrae were isolated after 4 weeks injection. Anti-human CD138+ mAb microbead was used to detect MM cells extracted from mice tissue by flow cytometry. Results Gene-expression profiling showed down-regulation of TNFRSFs, including TNFRSF11A, TNFRSF11B, TNFRSF8, TNFRSF10C, TNFRSF9, TNFRSF21, TNFRSF1B, TNFRSF1A and TNFRSF18, compared to normal plasma cells. Moreover, Our IHC results also showed that GITR expression was positive in primary CD138+ plasma cells from 9 normal bone marrow, but negative in 9 MM samples. Importantly, we found that low GITR expression significantly correlated with MM progression. Indeed, GITR gene levels were lower in smoldering and active MM patients compared to MGUS patients and normal donors. Promoter CpG island (CGI) methylation of GITR was indentified in 5 out of 7 MM primary bone marrow (BM)-derived CD138+ cells but not in normal BM-derived plasma cells. Bisulfate sequencing and Medip assay showed that methylation of GITR was significantly associated with GITR expression in 5 MM cell lines, including MM1.S, OPM1, U266, RPMI and INA6. Promoter CGI of GITR was highly methylated leading to complete silencing of GITR in MM1.S cell line. GITR expression was significantly up-regulated in MM cells upon treatment with the 5'azacytidine. MTT and BrdU assay revealed that the proliferation and survival of MM1.S cells was disrupted in the GITR over-expressing MM1.S cells, notably with inhibition of cell proliferation compared to control vector infected cells. Moreover induction of cytotoxicity in GITR over-expressing cells was confirmed by using GFP competition assay. GITR-induced apoptosis was supported by induction of caspase 8 and 3 cleavage. The inhibition of human CD138+ plasma cell growth in the bone marrow of SCID mice using a disseminated MM xenograft model was observed in the experimental group injected with GITR expressing cells compared to the control group after 4 weeks injection. Conclusion Our findings uncovered a novel epigenetic mechanism contributing to MM pathogenesis, showing the role of GITR methylation as a key regulator of MM cell survival. Disclosures: Roccaro: Roche:. Ghobrial:Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 1815 Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 (Raptor) and TORC2 (Rictor). TORC1 leads to the phosphorylation of p70S6 kinase and 4E- BP1, while TORC2 regulates phosphorylation of Akt and other kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin and its analogues have not shown significant activity in MM, likely due to the lack of inhibition of TORC2. In this study, we dissected the baseline activity of the PI3K/Akt/mTOR pathway TORC1/2 in MM cell lines with different genetic abnormalities. Methods: Eight different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, immunochemistry, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. In vivo homing was checked by in vivo flow. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: Raptor (TORC1) and Rictor (TORC2) knockdowns led to significant inhibition of proliferation of MM cells even in the presence of bone marrow stromal cells, this effect was also accompanied by inactivation of p-Akt, p-rS6 and p-4EBP1. We used INK128, a dual and selective TORC1/2 kinase inhibitor with similar effects to Raptor plus Rictor knockdown. We examined the protein expression levels of both mTOR complex and their downstream effectors in MM plasma cells from patients and cell lines. mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all samples. We showed that dual TORC1/2 inhibition is much more active than TORC1 inhibition alone (rapamycin) even in the presence of cytokines or stromal cells. INK128 induced cell cycle arrest, autophagy and apoptosis in cell lines and primary plasma cells even in the presence of bone marrow stromal cells (BMSCs). INK128 also showed a significant effect inhibiting cell adhesion in our in vivo homing model. Oral daily treatment with INK128 highly decreased the percentage of CD138+ tumor plasma cells in mice implanted with MM cells and reduced the levels of p-Akt and p-4EBP. These results suggest that potent and complete blockade of mTOR as part of TORC1 and TORC2 is potential therapeutic strategy to induce cell cycle arrest, apoptosis and disruption of MM cells interaction with the BM microenvironment. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Liu: Intellikine: Employment. Roccaro:Roche: Research Funding. Rommel:Intellikine: Employment. Ghobrial:Celgene: Consultancy; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Abstract 2728 Background. We and others have previously demonstrated that primary Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) cells show increased expression of microRNA-155 (miR-155), suggesting a role in regulating pathogenesis and tumor progression of these diseases. However, developing therapeutic agents that specifically target miRNAs has been hampered by the lack of appropriate delivery of small RNA inhibitors into tumor cells. We tested the effect of a novel LNA (locked nucleic acid)-modified anti-miR-155 in WM and CLL. Methods. WM and CLL cells, both cell lines (BCWM.1; MEC.1) and primary tumor cells; BCWM.1 Luc+ cells; and primary WM bone marrow (BM) stromal cells were used. WM and CLL cells were treated with antisense LNA anti-miR-155 or LNA scramble oligonucleotide. Efficiency of delivering FAM-labeled LNA into cells was determined by flow cytometry. Survival and cell proliferation were assessed by MTT and thymidine uptake assay, respectively. Synergistic effects of LNA with bortezomib were detected on BCWM.1 or MEC1 cells. Co-culture of BCWM.1 or MEC1 cells with WM bone marrow stromal cells was performed to better define the effect of the LNA-anti-miR155 in the context of the bone marrow microenvironment. miR-155 levels were detected in stromal cells from WM patients by qPCR. Co-culture of BCWM.1 or MEC1 cells with either wild-type or miR155−/− mice BM stromal cells was examined after LNA treatment. Gene expression profiling analysis was performed on BCWM.1 cells treated with either LNA anti-miR-155 or scramble control. miR-155 target gene candidates were predicted by TargetScan software. mRNA levels of miR-155, and its known target genes or gene candidates were detected by qRT-PCR. A microRNA luciferase reporter assay was used to determine whether miR-155 target candidates could be directly regulated by miR-155. mRNA levels of miR-155 targets were detected by qRT-PCR from primary WM or CLL cells treated with LNA. The activity of the LNA-anti-miR-155 was also detected in vivo using bioluminescence imaging and mRNA levels of miR-155 targets were detected by qRT-PCR ex vivo. Efficiency of introducing the FAM-labeled LNA into mice BM cells was determined by flow cytometry 1 week or 2 weeks after intravenous injection. Results. The efficiency of delivering LNA oligos into both WM and CLL-derived cell lines and primary samples was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines by 30–50%, as compared to LNA scramble control. In contrast, LNA antimiR-155 didn't exert significant cytotoxicity in BCWM.1 or MEC.1. LNA synergistically decreased BCWM.1 or MEC1 cell growth co-treated with bortezomib and decreased BCWM.1 or MEC1 cell growth co-cultured with WM BM stromal cells in vitro. A higher level of miR-155 was found in WM BM stromal cells compared to normal ones. LNA decreased BCWM.1 or MEC1 cell growth when co-cultured with BM stromal cells from miR155−/− mice compared with wild-type. We demonstrated increased expression of miR-155-known targeted genes, including CEBPβ, SOCS1, SMAD5, and several novel target candidates including MAFB, SH3PXD2A, and SHANK2, in WM cells upon LNA anti-miR-155 treatment. These target candidates were confirmed to be directly regulated by miR-155 using a luciferase reporter assay. mRNA levels of miR-155 targets were upregulated by 1.5–2 fold at 48 hr after direct incubation of the LNA with primary WM or CLL samples, indicating efficient delivery and biologic effect of the LNA in cells. Moreover, this LNA showed significant in vivo activity by inhibiting WM cell proliferation in a disseminated xenograft mouse model. Upregulation of miR-155 targeted genes were confirmed ex vivo, in WM cells isolated from the BM of treated mice compared to control. Mice BM cells were FAM positive 1 or 2 weeks after injection indicating efficient delivery of FAM-labeled LNA into cells in vivo. Summary. A novel LNA (locked nucleic acid)-modified anti-miR against miR-155 could be highly efficiently delivered into tumor cells in vivo in the bone marrow microenvironment. Anti-WM activity of LNA anti-miR-155 was confirmed both in vitro and in vivo and anti-CLL activity was confirmed in vitro. Novel miR-155 direct target genes including MAFB, SH3PXD2A, and SHANK2 were identified. These findings will help to design individualized clinical trials for WM and CLL patients with elevated levels of miR-155 in their tumor cells. Disclosures: Roccaro: Roche:. Obad:Santaris Pharma: Employment. Broom:Electroporation: Employment. Kauppinen:Santaris Pharma: Employment. Brown:Calistoga: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Genzyme: Research Funding; GSK: Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Membership on an entity's Board of Directors or advisory committees; Noxxon: Consultancy, Membership on an entity's Board of Directors or advisory committees; Millennium: Research Funding; Bristol-Myers Squibb: Research Funding; Noxxon: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 4914 Background. We and others have previously demonstrated that microRNA-155 (miR-155) is overexpressed in patients with Waldenstrom's Macroglobulinemia (WM) and Chronic lymphocytic leukemia (CLL) compared to healthy subjects; indicating that miR-155 may play a crucial role in regulating the pathogenesis of these diseases. We therefore evaluated the role of the novel locked nucleic acid (LNA) antimiR against miR-155 in WM and CLL-derived cell lines. Methods. WM and CLL-derived cell lines (BCWM.1, MEC1) have been treated with 20μM antisense LNA antimiR-155 or scramble probe (Santaris Pharma, Hørsholm, Denmark). Efficiency of delivered LNA oligos into cells was determined by using FAM-labeled LNA, followed by Flow cytometry (FACS). Survival and cell proliferation were assessed by MTT assay and thymidine uptake, respectively. Serum levels of miR-155 from peripheral blood of WM patients or healthy subjects and the level of miR-155-targeted genes were detected by quantitative RT-PCR (qPCR). Results. Efficiency of delivered LNA oligos into both WM and CLL-derived cell lines was higher than 90%. LNA antimiR-155 reduced proliferation of WM and CLL-derived cell lines, as compared to LNA scramble probe used as a control. In contrast, LNA antimiR-155 did not exert any effect on survival of them. We demonstrated increased expression of miR-155-targeted genes, such as CEBPβ, SOCS1, and TP53DINP1 in WM cells upon LNA antimiR-155 treatment. Moreover, we found miR-155 could be detected in peripheral blood serum of WM patients, but not the serum of healthy subjects. Summary. These data provide preclinical evidence for using the novel antisense LNA antimiR-155 in WM and CLL. Moreover, serum levels of miR-155 could potentially be used as a biomarker in patients with WM. Disclosures: No relevant conflicts of interest to declare.

Abstract 142 Introduction: Receptor tyrosine kinases (RTKs) are key regulators of the development and progression of many types of cancer. The Eph receptors represent the largest family of RTKs, which are used extensively throughout embryogenesis and development but detected in adult normal tissues. Elevated levels of expression of Eph receptors was found in a wide range of cancers and correlated with aggressive disease and metastasis. This is, triggered by a wide array of cellular responses, including cell boundary formation, motility, adhesion, and repulsion. Waldenstrom macroglobulinemia (WM) is characterized by widespread involvement of the bone marrow, indicating homing and adhesion of the malignant cells to specific niches in the bone marrow, which provides a protective environment for the survival and proliferation of these cells. Direct adhesion to endothelial cells and several growth factors and cytokines secreted by endothelial cells in the BM were shown to regulate tumor proliferation. In this study we aimed to characterize the role of RTKs, specifically Eph-B2 receptor, in the interaction of WM cells with the BM microenvironment including cell adhesion, proliferation, and cell-cycle. Methods and Results: We first examined the expression and activation (phosphorylation) of different RTKs in primary WM samples and cell lines using an antibody-based RTK-array. Eph-B2 receptor was activated in all patient samples compared to control, with a 5-fold increase in CD19+ WM cells, and we further confirmed that Eph-B2 was phosphorylated in WM cell lines, BCWM.1, and in IgM secreting cell lines RL, MEC-1. We next examined the activity of Ephrin-B2/Eph-B2 on adhesion and migration of WM cells. Treating starved WM cells with recombinant ephrin-B2 (the ligand of Eph-B2) activated cell-adhesion signaling, including focal adhesion kinase (FAK), Src, p130, paxilin and cofilin, but had no effect on WM cell chemotaxis. Moreover, we found that ephrin-B2 was highly expressed on endothelial cells isolated from the BM of WM patients and on HUVEC cells. Inhibition of ephrin-B2 on the endothelial cells or Eph-B2 on the WM cells with blocking antibodies inhibited the adhesion of the WM cells to endothelial cells, as well as the cytoskeletal signaling in WM cells induced by co-culture with endothelial cells. Consequently, inhibition of the ephrin-B2/Eph-B2 interaction reversed the proliferative and cell cycle effect in WM cell which was induced by co-culture with endothelial cells. This was confirmed using BrdU proliferation assay, flow cytometry for cell cycle, and by immuno-blotting to confirm the effect on proliferative signaling pathways including PI3K kinase and cell cycle related proteins including Cyclin-D, Cyclin-E and p-Rb. Conclusion: This study examines the interaction of Eph-B2 receptor in WM and shows that ephrin- B2/Eph-B2 axis regulates adhesion, activation of downstream signaling of integrin-related molecules, survival and cell cycle progression through the interaction of tumor cells with endothelial cells. This provides the basis for further studies to explore Eph-B2 as a novel therapeutic target in WM. Disclosures: Ghobrial: Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 3622 Introduction: Epigenetic factors such as DNA methylation have been shown to play a crucial role in the pathogenesis and progression of multiple myeloma (MM), yet studies of DNA methylation in MM are still limited. Therefore, in order to better understand the role of DNA methylation and identify specific genes that may be affected by differential methylation in MM patients, we conducted genome-wide DNA methylation profiling in cd138+ plasma cells purified from bone marrow of the patients with MM and normal donors. Methods: Genomic DNA of CD138+ Plasma cell selected from both MM patients and normal primary bone marrow was extracted using QIAGEN genome isolation kit. Following extraction, methylated DNA was isolated by Chip and hybridized to Affymetrix Human 2.0 tiling arrays. Chip assay and array hybridization was performed by Genepathway Inc. CEL files were processed and normalized using the MAT program, and methylation peaks were called from the resulting MAT scores using a custom segmentation method. Peak annotation and characterization of different genomic regions was done with custom tools and using genome annotation files from the UCSC genome database. All peaks were visualized by IGB online software. Medip-PCR was done in human MM cell lines to validate the methylation status. Methylated gene expression was determined by both Semi-quantitative PCR and real-time PCR. 5′aza was used for demethylation in human MM cell lines. Methylated gene expression with or without 5′aza treatment was determined by both Semi-quantitative PCR and real-time PCR. Results: Genomic DNA from CD138+ plasma cells from bone marrow of MM patients showed a significant increase in methylation levels compared to normal controls. We demonstrated that the hypermethylated sites were distributed across the genome in the following proportions: 3.2% in the promoter region; 45.6% in the intragenic region; 5.4 % in the 3′ end region; and 46.8 % in the intergenic region. Furthermore, around 9 % promoter CpG islands (CGIs); 11% intragenic CGIs; 15 % CGIs in 3′end region; and 14.3 % intergenic CGIs of patients genomic DNA were methylated. Moreover 2.1% promoter CGIs; 2.3 % intragenic CGIs; 2.5% CGIs in 3′end region; and 4.7% intergenic CGIs were methylated for the normal control. Medip-PCR showed that the identified methylation pattern in MM patients showed similar results in MM cell lines. Expectedly, we also observed that suppressor of cytokine signaling 1 (SOCS1) was hypermethylated at the promoter region (MAT score=19.986) as has been reported in human cell lines. Importantly, another member of SOCS family SOCS3 showed much stronger signal in the promoter region with CpG island (MAT score=31.707) in MM patients compared to normal control. Notably, the expression of two members of TNFR superfamily TNFRSF18 and TNFRSF4 which play an important role in development and programmed cell death of lymphocyte significantly have increased 283 and 141-fold after treatment with 5′aza in MM cell lines. Conclusion: These findings enhance our understanding of the role of DNA methylation in MM, as one of the epigenetic changes that may contribute to the pathogenesis of this disease. The identification and functional characterization of novel key molecules affected by DNA methylation will provide deeper insight into the molecular basis of MM disease. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Anderson:Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Abstract 3620 Introduction: Waldenstrom macroglobulinemia (WM) is a rare indolent non-Hodgkin lymphoma, characterized by bone marrow infiltration of clonal lymphoplasmacytic cells. Despite recent advances in understanding the pathogenesis of this disease, the molecular basis of WM etiology has not been clearly defined. We therefore performed genome-wide analysis of RNA polymerase II (pol II) binding sites and gene expression profiling in primary WM cells in order to comprehensively define the aberrant transcriptional regulation and related genes in WM. Methods: Primary CD19+ bone marrow derived WM cells and normal primary bone marrow were used. Genomic DNA was extracted using genome isolation kit (QIAGEN) after cross linking. All the DNA samples were sent for Chip assay and human promoter 1.0R array (Genepathway Inc.) which comprised of over 4.6 million probes tiled through over 25.500 human promoter regions. Each promoter region covers approximately 7.6kb upstream through 2.45kb downstream of the transcription start sites. For over 1,300 cancer associated genes, coverage of promoter regions was expanded to additional genomic content; for selected genes total coverage spans from 10kb upstream through 2.45kb downstream of transcription start sites. The published gene expression datasets (GDS2643) which included 10 CD19+ B cell from bone marrow of 10 WM patients and 8 normal controls was analyzed by d-chip software and normalized to normal control. The motif analysis was performed using Cistrome online tools from the Dana Farber Cancer Institute. The gene sets enrichment analysis (GSEA) was performed using GSEA online software from Broad institute. Results: A total of 13,546 high-confidence pol II sites were identified in WM samples and share a small percentage of overlap (11.5%) with the binding sites identified in normal controls. Combining the expression microarray data of WM patient samples and normal controls, we demonstrated a significant correlation between high levels of gene expression and enriched promoter binding of pol II. Notably, we also observed that the WM-unique pol II binding sites are localized in the promoters of 5,556 genes which are involved in important signaling pathways, such as Jak/STAT and MAPK pathways by applying gene set enrichment analysis (GSEA). Interestingly, we found that STAT, FOXO and IRF family binding sites motifs were enriched in the pol II-bound promoter region of IL-6 which plays a crucial role in cell proliferation and survival of WM cells. Moreover, the CpG island associated c-fos promoter was enriched for Pol II binding as compared to the normal control. Conclusion: The presence of increased Pol II binding and the identification of transcription factor motifs in the promoters of key oncogenes may lead to a better understanding of WM. Our findings suggest that altered transcriptional regulation may play an important role in the pathogenesis of WM. In addition, this study will provide novel insights into the molecular mechanism of WM etiology, and may lead to discovery of novel diagnostic molecular biomarkers and therapeutic targets for WM. Disclosures: Leleu: Celgene: Consultancy, Research Funding; Janssen Cilag: Consultancy, Research Funding; Leo Pharma: Consultancy; Amgen: Consultancy; Chugai: Research Funding; Roche: Consultancy, Research Funding; Novartis: Consultancy, Research Funding. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Abstract 133 Introduction: Mammalian target of rapamycin (mTOR) is a downstream serine/threonine kinase of the PI3K/Akt pathway that integrates signals from the tumor microenvironment such as cytokines and growth factors, nutrients and stresses to regulate multiple cellular processes, including translation, autophagy, metabolism, growth, motility and survival. Mechanistically, mTOR operates in two distinct multi-protein complexes, TORC1 and TORC2. Activation of TORC1 leads to the phosphorylation of p70S6 kinase and 4E-BP1, while activation of TORC2 regulates phosphorylation of Akt and other AGC kinases. In multiple myeloma (MM), PI3K/Akt plays an essential role enhancing cell growth and survival and is activated by the loss of the tumor suppressor gene PTEN and by the bone marrow microenvironment. Rapamycin analogues such as RAD001 and CCI-779 have been tested in clinical trials in MM. Their efficacy as single agents is modest, but when used in combination, they show higher responses. However, total inhibition of Akt and 4E-BP1 signaling requires inactivation of both complexes TORC1 and TORC2. Consequently, there is a need for novel inhibitors that can target mTOR in both signaling complexes. In this study we have evaluated the role of TORC1 and TORC2 in MM and the activity and mechanism of action of INK128, a novel, potent, selective and orally active small molecule TORC1/2 kinase inhibitor. Methods: Nine different MM cell lines and BM samples from MM patients were used in the study. The mechanism of action was investigated by MTT, Annexin V, cell cycle analysis, Western-blotting and siRNA assays. For the in vivo analyses, Luc+/GFP+ MM.1S cells (2 × 106/mouse) were injected into the tail vein of 30 SCID mice and tumor progression was detected by bioluminescence imaging. Nanofluidic proteomic immunoassays were performed in selected tumors. Results: To examine activation of the mTOR pathway in MM, we performed kinase activity assays and protein analyses of mTOR complexes and its downstream targets in nine MM cell lines. We found mTOR, Akt, pS6R and 4E-BP1 are constitutively activated in all cell lines tested independently of the status of Deptor, PTEN, and PI3K. All cell lines expressed either Raptor, Rictor or both; excepting H929 and U266LR7 which were negative for both of them. Moreover, primary plasma cells from several MM patients highly expressed pS6R while normal cells were negative for this protein. We found that INK128 and rapamycin effectively suppressed phosphorylation of p6SR, but only INK128 was able to decrease phosphorylation of 4E-BP1. We observed that INK128 fully suppressed cell viability in a dose and time dependent manner, but rapamycin reached a plateau in efficacy at ± 60%. The IC50 of INK128 was in the range of 7.5–30 nM in the eight cell lines tested. Similar results were observed in freshly isolated plasma cells from MM patients. Besides the induction of apoptosis and cell cycle arrest, INK128 was more potent than rapamycin to induce autophagy, and only INK128 was able to induce PARP and Caspases 3, 8 and 9 cleavage. In the bone marrow microenvironment context, INK128 inhibited the proliferation of MM cells and decreased the p4E-BP1 induction. Importantly, treatment with rapamycin under such conditions did not affect cell proliferation. INK128 also showed a significantly greater effect inhibiting cell adhesion to fibronectin OPM2 MM1S, BMSCs and HUVECs compared to rapamycin. These results were confirmed in vivo. Oral daily treatment of NK128 (1.0 mg/kg) decreased tumor growth and improved survival of mice implanted with MM1S. Conclusion: Dual inhibition of TORC1 and TORC2 represent a new and promising approach in the treatment of MM and its microenvironment. The ability of INK128 to inhibit both TORC1 and TORC2 strongly supports the potential use of this compound in MM patients. Disclosures: Anderson: Millennium Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Ghobrial:Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.